PROJECT SUMMARY/ABSTRACT ?1-adrenergic receptors (?1ARs) are the principle mediators of catecholamine actions in cardiomyocytes. ?1ARs rapidly adjust cardiac output by activating a Gs-adenylyl cyclase pathway that increases cAMP, activates protein kinase A, and phosphorylates substrates involved in excitation-contraction coupling. However, chronic ?1AR activation in the setting of heart failure leads to a spectrum of changes that contribute to adverse cardiac remodeling. This has been attributed to ?1AR activation of signaling pathways that induce cell death. While ?1ARs also activate signaling pathway that promote cell survival, this is not sufficient to afford cardioprotection in the setting of heart failure. The notion that ?1AR might contain specific molecular determinants that influence their signaling phenotype in cardiomyocytes (and influence the balance of signaling between pro-apoptotic vs. anti-apoptotic pathways) has never been considered. This application implicates the ?1AR extracellular N-terminus (which typically is dismissed as playing a negligible role in receptor activation/regulation mechanisms) as a heretofore-unrecognized structural determinant of ?1AR activation. We show that the ?1AR N-terminus is a target for O-glycosylation at two specific sites (S37/S41) adjacent to an N- terminal cleavage site, that O-glycosylation at these sites prevents ?1AR N-terminal cleavage, and that cleavage influences ?1AR signaling bias to cAMP/PKA vs. ERK pathways in cardiomyocytes. Studies in aim #1 of the application will take advantage of mutagenesis approaches (generating O-glycosylation-defective, truncated, and cleavage-resistant forms of the ?1AR) and a range of state-of-the-art cell-based approaches in cardiomyocytes (including studies with fluorescent sensors) to define the molecular basis for the altered signaling properties of O-glycosylation-defective/truncated ?1ARs in cardiomyocytes. The studies will focus on whether the N-terminus exerts its function as an inherent modifier of the ?1AR structure itself or whether the altered signaling phenotype of N-terminally truncated ?1ARs is due to changes in its subcellular trafficking/compartmentation pattern and/or coupling to signaling partners and downstream effectors. Aim #2 will use mouse models engineered to express mutant glycosylation defective/truncated or cleavage-resistant ?1ARs in place of the WT-?1AR, to determine the role of ?1AR N-terminal processing as regulator of cardiac function and catecholamine responsiveness. The overarching goal of these studies is to examine the role of the N-terminus as a novel structural determinant of the ?1AR that influences signaling to the proarrhythmic cAMP/PKA pathway, contributes to the pathologic cardiac remodeling, and can be pharmacologically targeted to prevent or mitigate the evolution of catecholamine-induced pathologic cardiac remodeling during heart failure.